Infused Fc-tagged beta-glucuronidase crosses the placenta and produces clearance of storage in utero in mucopolysaccharidosis VII mice.

Glycosaminoglycan storage begins in prenatal life in patients with mucopolysaccharidosis (MPS). In fact, prenatal hydrops is a common manifestation of MPS VII because of beta-glucuronidase (GUS) deficiency. One way to address prenatal storage might be to deliver the missing enzyme across the placenta into the fetal circulation. Maternal IgG is transported across the placenta by the neonatal Fc receptor (FcRn), which recognizes the Fc domain of IgG and mediates transcytosis from maternal to fetal circulation. We hypothesized that we could exploit this process to deliver corrective enzyme to the fetus. To test this hypothesis, the C-terminal fusion protein, GUS-Fc, was compared with native, untagged, recombinant GUS for clearance from the maternal circulation, delivery to the fetus, and reduction of lysosomal storage in offspring of MPS VII mice. We observed that GUS-Fc, infused into pregnant mothers on embryonic days 17 and 18, was transported across the placenta. Similarly infused untagged GUS was not delivered to the fetus. GUS-Fc plasma enzyme activity in newborn MPS VII mice was 1,000 times that seen after administration of untagged GUS and approximately 100 times that of untreated WT newborns. Reduced lysosomal storage in heart valves, liver, and spleen provided evidence that in utero enzyme replacement therapy with GUS-Fc targeted sites of storage in the MPS VII fetus. We hypothesize that this noninvasive approach could deliver the missing lysosomal enzyme to a fetus with any lysosomal storage disease. It might also provide a method for inducing immune tolerance to the missing enzyme or another foreign protein.

Nipped-A, the Tra1/TRRAP subunit of the Drosophila SAGA and Tip60 complexes, has multiple roles in Notch signaling during wing development.

The Notch receptor controls development by activating transcription of specific target genes in response to extracellular signals. The factors that control assembly of the Notch activator complex on target genes and its ability to activate transcription are not fully known. Here we show, through genetic and molecular analysis, that the Drosophila Nipped-A protein is required for activity of Notch and its coactivator protein, mastermind, during wing development. Nipped-A and mastermind also colocalize extensively on salivary gland polytene chromosomes, and reducing Nipped-A activity decreases mastermind binding. Nipped-A is the fly homologue of the yeast Tra1 and human TRRAP proteins and is a key component of both the SAGA and Tip60 (NuA4) chromatin-modifying complexes. We find that, like Nipped-A, the Ada2b component of SAGA and the domino subunit of Tip60 are also required for mastermind function during wing development. Based on these results, we propose that Nipped-A, through the action of the SAGA and Tip60 complexes, facilitates assembly of the Notch activator complex and target gene transcription.

Expression of His-tagged Shigella IpaC in Arabidopsis.

Although the expression of histidine (His)-tagged proteins in bacteria is routine, few His-tagged proteins have been expressed in plants, and no His-tagged proteins from bacterial pathogens have been expressed in plants, to our knowledge. Here, we demonstrate expression of the Shigella flexneri invasion plasmid antigen, IpaC, in Arabidopsis thaliana. S. flexneri is the causitive trigger for bacillary dysentery, and IpaC is essential for bacterial entry into epithelial cells. IpaC, attached to a 5' leader containing six tandem His codons, was cloned into a pBI121 vector. This clone was introduced into Agrobacterium tumefaciens and Arabidopsis plants were then transformed. T1 and T2 plant generations were obtained. Total plant proteins were extracted from T2 leaves; the Bradford assay was used to determine protein concentrations. A nickel-coated ELISA plate method, using both anti-His and anti-IpaC 1 degrees antibodies, was used to detect and quantify IpaC in transgenic Arabidopsis plants. Between 1.9 and 2.3 microg IpaC/mg total plant protein was obtained; this equals 0.2% of total protein, an amount comparable to other recombinant protein estimates in plants. Expressing His-tagged proteins from bacterial pathogens, in plants, is important because plant material could ultimately be fed or applied intranasally to animals that are "at risk" for infection by such bacterial pathogens, thus causing them to raise antibodies against the pathogens--functioning as a vaccine.

Maize Activator (AC) transposase (TPase) is expressed in Saccharomyces cerevisiae from a genomic clone. detection via Elisa, and proposed use in complementation studies.

The maize Activator (Ac) transposase (TPase) was expressed as a Histidine (His)-tagged protein in Saccharomyces cerevisiae from a full length genomic clone. Expression was demonstrated via the highly specific nickel-coated Elisa plate method, using an anti-His antibody and 2 separate anti-Ac TPase antibodies, to Ac residues 103-465 and 189-807. AC TPase expression in Saccharomyces is important for two reasons: (a) because the expression from a genomic clone herein permits the future study of RNA splicing mechanisms in common between maize and yeast systems, and (b) because a yeast system can easily be used for demonstrating complementation of function. Thus, such transformed yeast systems could be used in future, to experimentally test whether Ac TPase could complement various yeast mutations. Specifically, Ac TPase may be able to complement (i.e. provide the same function) to yeast transcription factor mutants or to genes mutated in other essential yeast functions. If confirmed, this would lend support to Barbara McClintock's hypothesis that transposable elements can serve as 'controlling elements' within the genome, by their ability to supplement other essential genes' functions, as needed. Work herein is contrasted with existing studies on Ac in yeast.

Maize Activator transposase expressed in Saccharomyces cerevisiae from a genomic clone: detection via Elisa, and proposed use in complementation studies.

The maize Activator (Ac) transposase (TPase) was expressed as a histidine (His)-tagged protein in Saccharomyces cerevisiae from a full-length genomic clone. Expression was demonstrated via the highly specific nickel-coated Elisa plate method, using an anti-His antibody and 2 separate anti-Ac TPase antibodies, to Ac residues 103-465 and 189-807. Ac TPase expression in S. cerevisiae is important for two reasons: (a) because the expression from a genomic clone herein permits the future study of RNA splicing mechanisms in common between maize and yeast systems, and (b) because a yeast system can easily be used for demonstrating complementation of function. Thus, such transformed yeast systems could be used in future to experimentally test whether Ac TPase could complement various yeast mutations. Specifically, Ac TPase may be able to complement (i.e., provide the same function) to yeast transcription factor mutants, or, to genes mutated in other essential yeast functions. If confirmed, this would lend support to Barbara McClintock's hypothesis that transposable elements can serve as 'controlling elements' within the genome, by their ability to supplement other essential genes' functions, as needed. Work herein is contrasted with existing studies on Ac in yeast.

Experimental evidence that the maize Activator (ac) transposase can act on a non-transposable element promoter to repress reporter gene expression in transient plant assays.

The transposase (TPase) encoded by the maize Activator (Ac) transposable element (TE) has been shown to repress the Ac TPase gene promoter (a form of element 'self-repression'). However, there is a lack of current experimental evidence to suggest that the Ac TPase can repress or activate the expression of non-TE gene promoters. Despite certain attributes of the maize Ac element and its TPase that might argue against such a broader, 'second function', that is, in regulating non-TE genes, the hypothesis that Ac TPase regulates gene expression was nevertheless tested in an in vivo tobacco BY-2 cell system using three different (non-TE) CaMV 35S promoters each linked to a gusA reporter gene. Four experiments were conducted; in each, BY-2 cells were co-transfected with two types of constructs: a CaMV 35S-gusA promoter construct (reporter) and an Ac expression construct (effector). In transient assays, a significant reduction in the magnitude of GUS enzyme activity was observed in the presence of an Ac effector (TPase-encoding) construct, below that observed for the 'no effector construct' treatment. A similar effector versus no effector comparison was made by Fridlender et al., who studied Ac element 'self-repression'. This Ac-associated GUS enzyme activity reduction was observed herein specifically for the gusA promoter construct with the 700 bp native CaMV 35S promoter region (a non-TE promoter). This construct contains 34 putative Ac TPase binding site motifs, 71% of which overlap one another on either DNA strand, motifs which also fall into the class of 'CpG islands' in plants. It is suggested that the Ac TPase can bind to such Ac DNA motifs and CpG islands existing within non-TE promoter regions, and can function as a regulator of transcription. Such a transcriptional mode of regulation has not been shown explicitly herein. The implications of the findings are discussed and a genomic (whole genome) regulation model is presented.